Abstract
摘要：角膜發炎經常伴隨著新生血管的形成和角膜混濁。因為角膜透明度對清晰的視力極為重要、角膜一旦產生混濁或疤痕將造成視力減退。架構蛋白在控制細胞骨架的組成中扮演重要的角色。 IQGAP1 便是一種具備多樣細胞功能的架構蛋白。IQGAP1 能影響細胞粘連、移動、入侵、增殖和血管的生成。先前有關IQGAP1 之研究均與癌症相關，而IQGAP1 在人類角膜上皮細胞的角色目前尚未有任何發表。在此綜合性研究中，我們希望建立IQGAP1 在綠膿桿菌角膜炎的致病機轉中之重要性，並探討由隱形眼鏡所導致之缺氧性新生血管和化學外傷所引發的新生血管中IQGAP1 扮演的角色。此研究的第一部份希望探討IQGAP1 在綠膿桿菌角膜炎特別是侵入基因型綠膿桿菌菌株的入侵機轉。我們過去的研究顯示由細菌所表現的第三類分泌系統(Type III secretion system，T3S)之毒素而分類的侵入基因型綠膿桿菌菌株對抗生素的敏感性差且治療後仍易造成嚴重的視力損害。因此，對T3S 系統侵入型菌株入侵機制的瞭解極為重要。我們初步的實驗結果發現人類角膜上皮細胞正常會表現IQGAP1 蛋白。IQGAP1 蛋白的表現主要在細胞膜的邊緣和細胞核周圍的區域。 IQGAP1 和上皮黏合交接(adherensjunction)蛋白中的E-cadherin 會在相同位置表現。角膜上皮細胞被侵入型綠膿桿菌菌株感染後，細胞膜的邊緣會產生板狀偽足(lamellipodia)或膜伸展(membrane extensions)之凸起，而且這些細胞膜型態的改變常伴隨有綠膿桿菌聚集在細胞膜偽足附近也同時與IQGAP1 蛋白聚集的位置相呼應。共軛焦螢光顯微鏡進一步顯示IQGAP1 蛋白會從原本在細胞邊緣的位置改到聚集在細胞膜下之細胞質內的區域進行重組。以Gentamicin 侵入試驗觀察侵入型菌株感染IQGAP1 敲減(knockdown)後的角膜上皮細胞之表現，我們發現IQGAP1 被敲減後，侵入型綠膿桿菌菌株侵入至細胞內的量銳減。在感染兩小時後的IQGAP1 敲減細胞內，細菌的侵入量明顯減少約八成。這些初步結果顯現IQGAP1 確實會影響綠膿桿菌的入侵。IQGAP1 的表現以敲減方式降低時可以相對的提供人類角膜上皮細胞對抗綠膿桿菌感染的保護作用。由於以上的結果，我們希望進一步了解IQGAP1 對於細胞交接蛋白(junctional proteins)及傷口修復中的角色以奠定IQGAP1 作為未來可用於治療目標的基礎。在研究的第二部分，我們希望進一步拓展對血管形成和血管角膜侵入的機制。由我們先前的研究顯示，綠膿桿菌角膜炎多發生於配戴隱形眼鏡者。然而，大部份配戴隱形眼鏡的人在發生角膜感染前並無明顯的症狀。由於隱形眼鏡可能經由角膜缺氧而誘發新生血管導致細微的眼表面變化，我們因此希望藉由我們已建立的兔子缺氧新生血管動物實驗模式來研究引發血管生成的初期機轉。另外也將以化學傷害所引發的兔子角膜新生血管實驗中，進一步比較與分析新生血管生成的機轉是否與IQGAP1 有關。本研究最終的部份將以IQGAP1敲減的方式降低此蛋白在角膜上皮細胞中的表現作為治療的目標。我們將在不同的動物試驗中比較綠膿桿感染性角膜炎、缺氧性隱形眼鏡和化學外傷新生血管對局部siRNA 治療的效果。總結:由初步實驗結果可知IQGAP1 與促使綠膿桿菌入侵有關，在綜合此三大部份的研究後，我們希望能更進一步剖析並釐清IQGAP1 在角膜感染疾病和新生血管生成中的角色並提供未來治療與改善臨床預後的新方向。
Abstract: Corneal inflammations are frequently accompanied by neovascularizations and corneal opacification thatpermanently affect vision. Scaffold proteins play important roles in controlling cell cytoskeletonorganization. IQ-domain GTPase-activating protein1 (IQGAP1) is one such protein with diverse cellularfunctions affecting cellular adhesions, migrations, invasion, proliferation, and angiogenesis. Previousunderstanding of IQGAP1 has been mostly through cancer related research. As far as we know, its role inthe human corneal epithelial (HCE) cell has not yet been reported. In this comprehensive research, wehope to establish IQGAP1 as a significant contributor in the pathogenesis of P. aeruginosa keratitis andneovascularizations due to contact lens induced hypoxia and chemical injury. The first part of our researchinvolves determining the role of IQGAP1 in the pathogenesis of P. aeruginosa keratitis. We havepreviously shown that P. aeruginosa strains with invasive genotype of Type III secretion (T3S) system issignificantly associated with poor antibiotic sensitivity and detrimental visual loss. We are thusespecially interested in the invasion mechanisms of T3S system invasive P. aeruginosa strains. Ourpreliminary data shows that HCE cells normally express IQGAP1. The localization of IQGAP1 is mainlyat cellular borders and perinuclear region. Colocalization of IQGAP1 with E-cadherin, an adherensjunction protein, is also demonstrated. Upon infection by invasive T3S system strains, lamellipodia ormembrane extensions appear with colocalization of P. aeruginosa and IQGAP1. Confocal microscopyfurther shows reorganization of IQGAP1 protein to subcellular cytoplasmic regions. Quantitativeanalysis of P. aeruginosa invasion into IQGAP1 knockdown HCE cells done by gentamicin invasion assayshowed a significant decrease in intracellular invasion at 2 hours post infection. Up to 80% decrease inintracellular invasion is noted. These initial results indicate that IQGAP1 plays a significant role inaffecting the invasion of P. aeruginosa. Knockdown of IQGAP1 in HCE cells gives a relative protectiveeffect against pseudomonal infection. With these promising background, we hope to further investigateIQGAP1’s involvement in affecting junctional proteins and wound healing to provide a basis for targetingIAGAP1 as a viable treatment option. As a second part of this research, we hope to expand our researchfurther into understanding the mechanisms involved in promoting blood vessel formation and invasion intothe corneal tissue. As our previous research have shown, P. aeruginosa keratitis is most commonlyassociated with contact lens wear. However, most contact lens wearers are asymptomatic beforedeveloping frank clinical symptoms of corneal infection. We are thus interested in determining thepossible effect of hypoxia due to contact lenses that may induce early ocular surfaces changes related toneovascularization formation through a rabbit hypoxia induced corneal neovascularization that we haddeveloped. We also would like to understand if the initiation of angiogenesis involves IQGAP1 inchemical injury rabbit models. As a last part of our research, application of siRNA to knockdownIQGAP1 in our pseudomonal infection, contact lens hypoxia, and chemical injury animal models willprovide clinical implications for this research. In summary, we have shown evidence that IQGAP1 ispossibly involved in promoting P. aeruginosa invasion. Further in-depth research in the role of IQGAP1in both infection and angiogenesis will provide possible therapeutic targets to improve clinical outcome.
Keyword(s)
架構蛋白IQGAP1
第三類分泌系統
綠膿桿菌
新生血管
缺氧
Scaffold protein IQGAP1
Type III Secretion System
P
aeruginosa
Neovascularizations
Hypoxia
